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Characterization of Francisella Species Isolated from the Cooling Water of an Air Conditioning System Brazilian Journal of Microbiology 46, 3, 921-927 (2015) Copyright © 2015, Sociedade Brasileira de Microbiologia ISSN 1678-4405 www.sbmicrobiologia.org.br DOI: http://dx.doi.org/10.1590/S1517-838246320140465 Research Paper Characterization of Francisella species isolated from the cooling water of an air conditioning system Quan Gu1, Xunde Li3, Pinghua Qu2, Shuiping Hou1, Juntao Li1, Edward R. Atwill3, Shouyi Chen1 1Guangzhou Center for Disease Control and Prevention, Guangzhou, China. 2Guangdong Hospital of Traditional Chinese Medicine, University City Branch, Guangzhou, China. 3Department of Population Health and Reproduction, University of California, California, USA. Submitted: May 30, 2014; Approved: December 28, 2014. Abstract Strains of Francisella spp. were isolated from cooling water from an air conditioning system in Guangzhou, China. These strains are Gram negative, coccobacilli, non-motile, oxidase negative, catalase negative, esterase and lipid esterase positive. In addition, these bacteria grow on cysteine- supplemented media at 20 °C to 40 °C with an optimal growth temperature of 30 °C. Analysis of 16S rRNA gene sequences revealed that these strains belong to the genus Francisella. Biochemical tests and phylogenetic and BLAST analyses of 16S rRNA, rpoB and sdhA genes indicated that one strain was very similar to Francisella philomiragia and that the other strains were identical or highly simi- lar to the Francisella guangzhouensis sp. nov. strain 08HL01032 we previously described. Biochem- ical and molecular characteristics of these strains demonstrated that multiple Francisella species exist in air conditioning systems. Key words: Francisella, air conditioning, water. been utilized in phylogenetic analysis of Francisella Introduction (Barns et al., 2005). It is recognized that the aerosol generated by air con- Francisella is a genus of Gram-negative, coccoba- ditioning systems is one medium of transmission of respira- cilli, non-motile and aerobic bacteria (Pechous et al., tory pathogens, such as Legionella pneumophila (Stout and 2009). Currently, five species have been recognized in the Yu, 1997). There is a knowledge gap regarding the exis- genus: F. tularensis, F. philomiragia, F. noatunensis, F. tence of bacterial species in the cooling water of air condi- hispaniensis and F. halioticida (Brevik et al., 2011; Fran- tioning systems. In 2008, we isolated the first strain of cis, 1922; Hollis et al., 1989; Huber et al., 2009; Ottem et Francisella sp. (ID 08HL01032) (NCTC 13503) from the al., 2009). F. tularensis was the first Francisella species to cooling water of an air conditioning system in Guangzhou, be discovered in 1912 in the USA (McCoy et al., 1912) and China (Qu et al., 2009), which was the first report of is the pathogen that causes tularemia in the northern hemi- Francisella spp. isolated from air conditioning cooling wa- sphere. The other species were frequently isolated in the en- ter. This strain was named Francisella guangzhouensis sp. vironment or in marine fish with unknown pathogenicity to nov. (Qu et al., 2013). Between 2009 and 2011, nine addi- humans. One of the most common methods for identifica- tional Francisella spp. strains were isolated from the cool- tion of Francisella species is 16S rRNA gene sequencing ing water of air condition systems in the same city. Herein, (Barns et al., 2005; Kreizinger et al., 2013; Sjöstedt et al., we describe the biochemical characteristics and multiple 1997). In addition to the 16S rRNA gene, some housekeep- gene fingerprinting of these strains, which demonstrate the ing genes, such as the RNA polymerase subunit b (rpoB) presence of multiple species of Francisella and the consis- gene and the succinatede hydrogenase A (sdhA) gene, have tent presence of Francisella guangzhouensis sp. nov. in the Send correspondence to S. Chen. Department of Parasitic and Endemic Disease, Guangzhou Center for Disease Control and Prevention, 510440 Guangzhou, China. E-mail: [email protected]. 922 Gu et al. cooling water of air conditioning systems in Guangzhou, biochemical tests performed using the three kits and the ob- China. tained results. Materials and Methods Molecular analysis DNA was extracted from bacteria using a Qiagen Isolation of bacteria DNA mini kit according to the manufacturer’s manual. DNA concentrations were measured by a spectrophoto- A total of 312 water samples were collected from meter at A260/A280, and those with concentrations be- cooling towers of air conditioning systems in metro stations tween 1.7 and 2.1 were used as templates for PCR to and hotels in Guangzhou, China and were used for the de- amplify 16S rRNA, rpoB, and sdhA genes. For PCR of the tection of bacterial pathogens. Samples were collected be- 16S rRNA gene, approximately 1500 bp were amplified us- tween 2009 and 2011 as a part of the city’s continuous ing the primers 27F and 1492R, as described by Lane surveillance program conducted by the Guangzhou Center m for Disease Control and Prevention (GZCDC). Samples (1991). Each 50 L PCR reaction mixture included 10 mM were stored at 4 °C and processed in less than 3 days after Tris (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM sampling. Previously described methods were used for iso- deoxynucleoside triphosphates, 0.1 M of each primer, m lation of Francisella spp. (Qu et al., 2009). Briefly, water 2.5 U Taq (TAKARA) and 1 g of genome DNA template. samples (250 mL each) were filtered through 0.22 mm PCR cycles consisted of denaturation at 94 °C for 4 min, polycarbonate filters. Filters with filtrate were suspended in followed by 35 cycles of 94 °C for 30 s, 65 °C for 40 s, and 5.0 mL of sterilized water in a 50 mL centrifuge tube and 72 °C for 90 s, and extension at 72 °C for 5 min. For PCR of treated with an equal volume of acid buffer (pH 2.2) for the rpoB gene and sdhA gene, the primers used were RL1 5 min at room temperature. Then, 0.1 mL of the acidified and RL2 for the rpoB gene (Ko et al., 2002) and sdhF and sdhR for the sdhA gene (Barns et al., 2005). The PCR reac- suspension was placed onto Legionella-BCYEa-agar or tion mixture and conditions for both of these genes were BCYEa-GVPC (BCYEa supplemented with 3 g glycine identical to those used for the 16S rRNA gene. PCR cycles L-1, 80,000 IU polymyxin B sulfate L-1, 1 mg vancomycin consisted of denaturation at 94 °C for 4 min, followed by L-1 and 80 mg cycloheximide L-1) and incubated for 7 days 35 cycles of 94 °C for 30 s, 50 °C for 40 s, and 72 °C for 90 at 30 °C under an atmosphere of 5% CO . Suspected colo- 2 s, and extension at 72 °C for 5 min. The rpoB primers am- a nies were inoculated onto BCYE cysteine heart agar sup- plified a 330 bp product, and the sdhA primers amplified a plemented with 9% heated (chocolatized) sheep red blood 350 bp product. For all PCR experiments, DNA extracted cells (CHAB) and Columbia media agar, respectively, and from F. philomiragia (ATCC 25015) was used as positive incubated for 5 days under the same conditions as above. control and sterilized water was used as negative control. a The BCYE and CHAB media were cysteine supple- PCR products were verified by electrophoresis on 2% mented and suitable for the growth of Francisella spp. agarose gel stained with ethidium bromide. (Petersen et al., 2009). The PCR products of 16S rRNA, rpoB and sdhA genes were sequenced by the Invitrogen Laboratory in Phenotypic characteristics Guangzhou, China, using the same primers as above. DNA Bacterial colonies were isolated from BCYEa plates sequences were analyzed using the SeqScanner 1.0 soft- after 3-5 days of cultivation. Colonies of each strain were ware, and the bases with a QV value less than 20 were de- stained with Gram stain and examined with a bright field leted. Sequences were analyzed by multiple alignment microscope (1000x). To determine the most suitable analysis using Clustal X (Goujon et al., 2010). Sequences growth temperatures, a colony of each strain was inocu- of currently available Francisella type species in GenBank lated into brain heart infusion broth (BHI) with L-cysteine were used to construct phylogenetic trees that were inferred and incubated at 20, 25, 30, 35 and 40 °C for 12 h (Qu et al., using neighbor-joining analysis by the MEGA 5.0 software 2009). The bacterial concentrations after incubation at each (Tamura et al., 2011). Bootstrap values were determined temperature were measured by a spectrophotometer at using 1,000 replicates, and Legionella pneumophila was 600 nm. used as out-group. For each gene, sequences were analyzed to match the maximal identical sequence by BLAST analy- Biochemical characteristics sis using NCBI’s online BLAST tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Colonies isolated from BCYEa plate after 3 days of cultivation were used for biochemical tests. Oxidase and Results and Discussion catalase activity were tested using a BioMerieux API 20STREP kit and Vitek 2 (GN card) kit (BioMerieux). Nine strains of Francisella spp. bacteria (coded GZ- Other biochemical and enzymatic activities were tested by 01 to GZ-09) were isolated from 312 air conditioning cool- using API ZYM (BioMérieux) kit. Table 1 shows all of the ing water locations in the city of Guangzhou. The 9 strains Francisella in air conditioning water 923 Table 1 - Comparison of biochemical characteristics of Francisella sp. strains, F. guangzhouensis sp. nov. (NCTC 13503), and F. philomiragia (ATCC25015). Biochemical test Francisella spp.a Francisella sp. (GZ-04) F. philomiragia F. guangzhouensis sp. nov. (ATCC25015) (NCTC 13503) b Oxidase - - + - Catalase W - W W Urease - - - - Cysteine for growth + + - + PYRA - + + - aGAL -+ + - bGUR -+ + - bGAL -+ + - PAL - + + - LAP - + + - RIB + + + + Alkaline phosphatase - + + - Esterase + + + + Esterase lipase + + + + Leucine arylamidase - + + - Valine arylamidase - + + - Acid phosphatase + + + + Naphthol phosphohydrolase + + + + a-galactosidase -+ + - b-galactosidase -+ + - Chitinase - + + - aFrancisella sp.
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